In vitro synthesis of recombinant heparan sulfate

说明(申请人提供)：硫酸乙酰肝素(HS)是一种重要的多糖，大量存在于细胞表面和细胞外基质中。HS参与多种生理和病理生理功能，包括凝血、炎症反应和细胞分化。HS是一种高度硫酸盐化的多糖。肝素是HS的一种特殊形式，是一种常用的抗凝药物。HS广泛的生物学功能引起了人们对开发肝素或类肝素分子来开发抗癌和抗病毒药物的极大兴趣。HS独特的硫酸盐化模式被认为调节其功能特异性。然而，HS的化学合成是极其困难的，尤其是那些比六糖更大的化合物。使用HS生物合成酶，我们的实验室可以生产出一系列具有独特的硫酸盐化模式和功能的HS。我们的成功证明了以酶为基础合成具有独特生物活性的HS的可行性。该项目的长期目标是开发一种合成具有高结构精度的HS的方法。我们的假设是，N-磺基葡萄糖残基的分布决定了硫酸乙酰肝素生物合成过程中所有后续修饰的敏感性，包括异构化、2-O-硫化、6-O-硫化和3-O-硫化。在这项提议中，我们将使用糖基转移酶合成携带N-磺基葡萄糖胺残基的结构定义的寡糖。然后，我们将确定各种HS生物合成酶的底物特异性，目的是在寡糖中精确定位6-O-磺基和3-O-磺基以及2-O-磺基艾杜糖醛酸。最后，我们计划利用这种方法来研究新型抗凝血剂HS结构。该项目的成功将引领一种全面的新方法来研究HS的结构和功能关系，并可能导致基于HS的新型治疗试剂的开发。与公共卫生相关：硫酸乙酰肝素(HS)是一种高度硫酸盐化的多糖。肝素是HS的一种特殊形式，是一种常用的抗凝药物。HS广泛的生物学功能引起了人们对开发肝素或类肝素分子来开发抗癌和抗病毒药物的极大兴趣。HS独特的硫酸盐化模式被认为调节其功能特异性。该项目的长期目标是开发一种合成具有高结构精度的HS的方法。该项目的成功将引领一种全面的新方法来研究HS的结构和功能关系，并可能导致基于HS的新型治疗试剂的开发。

DESCRIPTION (provided by applicant): Heparan sulfate (HS) is an essential glycan that is present in large quantities on the cell surface and in the extracellular matrix. HS participates in a variety of physiological and pathophysiological functions, including blood coagulation, inflammatory response and cell differentiation. HS is a highly sulfated polysaccharide. Heparin, a special form of HS, is a commonly used anticoagulant drug. The wide range of biological functions of HS attracts considerable interest in exploiting heparin or heparin-like molecules for the development of anticancer and antiviral drugs. The uniquely distributed sulfation pattern of HS is believed to regulate its functional specificity. However, chemical synthesis of HS, especially those larger than hexasaccharides, is extremely difficult. Using HS biosynthetic enzymes, our labs can produce an array of HS with unique sulfation patterns and functions. Our success has proved the feasibility of conducting enzyme-based synthesis of HS with unique biological activities. The long term aim of this project is to develop a method to synthesize HS with high structural precision. Our hypothesis is that the distribution of N- sulfoglucosamine residues determines the susceptibility of all subsequent modifications during heparan sulfate biosynthesis, including epimerization, 2-O-sulfation, 6-O- sulfation, and 3-O-sulfation. In this proposal, we will synthesize structurally defined oligosaccharides carrying N-sulfoglucosamine residues using glycosyl transferases. We will then determine the substrate specificities of a variety of HS biosynthetic enzymes with the aim of precisely positioning the 6-O-sulfo and 3-O-sulfo groups as well as 2-O- sulfo iduronic acid within oligosaccharides. Finally, we plan to utilize this method to investigate novel anticoagulant HS structures. The success of this project will lead a comprehensive new approach to investigate the structure and function relationship of HS, potentially leading to the development of novel HS-based therapeutic reagents. Public Health Relevance: Heparan sulfate (HS) is a highly sulfated polysaccharide. Heparin, a special form of HS, is a commonly used anticoagulant drug. The wide range of biological functions of HS attracts considerable interest in exploiting heparin or heparin-like molecules for the development of anticancer and antiviral drugs. The uniquely distributed sulfation pattern of HS is believed to regulate its functional specificity. The long term aim of this project is to develop a method to synthesize HS with high structural precision. The success of this project will lead a comprehensive new approach to investigate the structure and function relationship of HS, potentially leading to the development of novel HS-based therapeutic reagents.